The bifunctional aminoglycoside-modifying enzyme AAC(6')-Ie-APH(2)-Ia is the single most important resistance enzyme for aminoglycoside antibiotics in Gram-positive cocci as it is singlehandedly produces resistance to virtually all aminoglycoside antibiotics and broadly disseminated among clinical enterococcal and staphylococcal isolates. Appearance of the closely-related monofunctional APH(2)-Ib phosphotransferase and AAC(6')-Im acetyltransferase in Gram-negative bacteria heralding the potential for spread of these expanded- spectrum enzymes among a wide variety of clinically important pathogens. Lack of kinetic and structural information regarding these enzymes constitutes the major impediment for the design of novel aminoglycoside antibiotics and inhibitors of aminoglycoside-modifying enzymes. The long-term goal is to delineate kinetic and structural mechanisms of interaction of aminoglycoside-modifying enzymes with their substrates to provide strong basis for the rational design of new effective antibiotics. The objectives in this particular application is to conduct detailed studies of the APH(2)-Ib, AAC(6')-Im, and AAC(6')-Ie-APH(2)-Ia enzymes by pursuing three specific aims: 1. Perform kinetics and structural studies of the AAC(6')-Im, APH(2)-Ib, and AAC(6')-Ie- APH(2)-Ia aminoglycoside-modifying enzymes; 2) Elucidate molecular mechanisms for the nucleotide triphosphate selectivity by the aminoglycoside phosphotransferases; and 3) Evaluate evolutionary potential of the bifunctional AAC(6')-Ie-APH(2)-Ia aminoglycoside-modifying enzyme. Multidisciplinary approach that include molecular biology, protein chemistry, detailed enzymology, purification and structural analyses of the products of the reactions by NMR and mass spectrometry, and X-ray crystallography of the native enzymes and their complexes with products, substrates and substrate analogues will be utilized to elucidate mechanistic aspects of these clinically important antibiotic-resistance enzymes. The detailed structural and kinetic studies of the AAC(6')-Im, APH(2)-Ib, and AAC(6')-Ie-APH(2)-Ia enzymes and elucidation of the molecular mechanism of recognition of nucleotide triphosphates (NTPs) by aminoglycoside phosphotransferases disclosed in this grant proposal will provide insight into their unique over-encompassing breadth of activity. These studies will also contribute to our understanding of the mechanisms of these enzymes, and provide solid basis for the rational design of novel aminoglycoside antibiotics resistant to modification by the aminoglycoside-modifying enzymes and inhibitors targeting the nucleotide binding site of aminoglycoside phosphotransferases. Evaluation of the evolutionary potential of the bifunctional AAC(6')-Ie-APH(2)-Ia enzyme towards broadening the spectrum of conferred resistance to aminoglycoside antibiotics would provide an important guidance for the future utility of clinically important aminoglycosides. Ultimately, such knowledge will provide the basis for the design of new aminoglycoside antibiotics and inhibitors of the aminoglycoside- modifying enzymes for treatment of life-threatening infections.